Vectors and methods for regenerative therapy

ABSTRACT

An expression vector capable of disrupting the silencing of cell cycle genes in adult cells, such as adult cardiac myocytes and other quiescent cells in terminally differentiated tissues, comprising: (a) a nucleic acid sequence encoding lysine-specific demethylase 4D (KDM4D); (b) a promoter that induces or effects overexpression of KDM4D, wherein the promoter is operably linked to the nucleic acid sequence; and (c) a regulatory element that inducibly represses the overexpression of KDM4D. The vector can be administered to a subject in a method for inducing tissue-specific hyperplasia in a mammal, including cardiomyocyte proliferation. The method provides for regenerative therapy, including improving cardiac function after myocardial infarct and other forms of cardiac damage.

This application claims benefit of U.S. provisional patent applicationNo. 62/150,159, filed Apr. 20, 2015, the entire contents of which areincorporated by reference into this application.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. R01HL070748, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The content of the ASCII text file of the sequence listing named“UW57WOU1_ST25”, which is 15 kb in size was created on Apr. 19, 2016,and electronically submitted via EFS-Web with this application isincorporated herein by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to nucleic acid molecules, vectors, cells,and related compositions and their use for inducing proliferation ofquiescent cells and in methods of regenerative therapy.

BACKGROUND OF THE INVENTION

The vast majority of mammalian cardiac myocytes (CM) stop proliferatingsoon after birth and subsequent heart growth predominately comes fromhypertrophy, an increase in cell size, instead of hyperplasia, anincrease in cell number. Because CM proliferation is required for theheart regeneration seen in lower vertebrates and neonatal mammalianinjury models, there is great interest in understanding the mechanismsregulating CM cell cycle exit and whether this cell cycle withdrawal canbe reversed.

Ischemic heart disease leading to heart failure^(1, 2) is the leadingcause of death in the world³. Although adult human hearts are unable toreplace lost CMs after injury, substantial cardiac regeneration is seenin lower vertebrate and mammalian models. Adult zebrafish⁴ and neonatalmice are able to regenerate their hearts after ≥15% has been amputated.Models of myocardial infarction (MI) in newborn mice offer a moreclinically relevant injury model to demonstrate heart regenerationcapacity in mammals^(6, 7). A common finding in these studies was themechanism by which cardiac regeneration occurred. Blood clot formation,inflammation, and collagen deposition were seen in response to theinjuries, but ultimately new CMs repopulated the lost tissue. Fatemapping studies revealed that the new cardiac myocytes came fromdedifferentiation and proliferation of pre-existing cardiac myocytes, incontrast to cardiac progenitor or stern cells⁴⁻⁶. However, when cardiacinjury was induced in mice at a later time-point, postnatal day 7 (P7),the regenerative response was lost leading to fibrotic scarring^(5, 6)similar to what is seen with human MIs^(1, 2). Thus, mammalian heartslose their regenerative capacity early in life, a process that requiresCM proliferation.

The poor regenerative response seen in adult hearts highlights thequestion of whether there is any turnover of CMs in adults. Though therehas been controversy over the extent of ACM proliferation, elegantstudies have estimated a 0.8% annual renewal rate in mammals^(8, 9). Butthis very limited source of new ACMs was demonstrated to come frompre-existing ACMs⁸. The rate of ACM hyperplasia increased slightly afterMI, though most DNA-synthesis activity resulted in polyploidization andmulti-nucleation, rather than complete cell division⁸. Consistent withvery rare ACM cell division, gene expression analysis reveals a dramaticdownregulation of cell cycle progression genes in ACMs compared withembryonic CMs¹⁰. Pressure-overload trans-aortic-constriction (TAC)models stimulate ACM expression of G1/S-phase promoting genes, but genesthat promote mitosis and cytokinesis remain silenced¹⁰. As G1/S-phasegenes are required for CM hypertrophyl^(11, 12), the gene expressionresults are consistent with the hypertrophy-restricted growth andincreased DNA-content displayed in ACMs after TAC or MI^(8, 13). Thus,ACMs have cell growth that is uncoupled from cell division¹⁴.Interestingly, CM switching to hypertrophic growth coincides with thepostnatal loss of regeneration capacity^(5, 6, 13).

The stable silencing of G2/M and cytokinesis genes represents part ofthe change in gene expression profile that occurs when CMs undergoterminal differentiation¹³. Recent studies suggest epigeneticmechanisms, such as post-translational modifications of histoneproteins, DNA methylation, and non-coding RNAs, direct the changes ingene expression that occur during cardiac development anddisease^(6, 15-19). Modulating epigenetic mechanisms can delay CM lossof proliferative and regenerative potential until adolescence, butregeneration in adult hearts remains elusive²⁰. Simplistically, thereare two types of epigenetically-defined chromatin structure andfunction: accessible and actively transcribed euchromatin, and, incontrast, condensed and transcriptionally silencedheterochromatin^(21, 22). Each chromatin type is associated withdistinct sets of histone modifications and chromatin-associatedproteins²²⁻²⁴. Histone modifications are thought to establish differentstates of chromatin by physically altering its structure²⁵⁻²⁷, as wellas recruiting other effector proteins which possessmodification-specific-binding domains^(21, 22). In general, euchromatinis enriched with histone acetylations, H3K4me3, and H3K36me3, whichrecruit transcriptional machinery²². In contrast, heterochromatin isenriched with H3K9me3, H3K27me3, and H4K20me3: repressive methylationsthat recruit heterochromatin-protein-1 (HP1) family members, Polycombproteins, and other repressive effectors^(21, 22). Interestingly, cellsthat have permanently exited the cell cycle show a striking differencein the organization of chromatin within the nucleus. In proliferatingfetal CMs, there is limited heterochromatin that is organized into manysmall foci within the nucleus, while in ACM, these foci accumulate intofew, large foci with additional heterochromatin at the nuclear lamina¹⁰.Similar patterns are observed in other non-proliferative cells;accumulation of heterochromatin coincides with terminal differentiationand cell cycle-exiting²⁸⁻³⁰.

E2F and Retinoblastoma family members (Rb, p107, p130) are at theinterface of cell cycle gene and chromatin structural regulation³¹⁻³⁴.In proliferating cells, E2F family proteins bind to a consensus sequencefound in the promoters of many cell cycle progression genes, acting asmaster regulators of cell division^(34, 35). When hypophosphorylated Rbfamily members bind to E2Fs, they inhibit cell cycle gene expression andcell proliferation. However, mitogenic stimulation can lead tophosphorylation of Rb proteins, freeing E2Fs to activate cell cycle geneexpression³⁴. In contrast to quiescent cells, terminally differentiatedskeletal and cardiac myocytes do not proliferate in response tomitogenic stimuli^(36, 37). This permanent cell cycle exit is mediatedby Rb-dependent recruitment of H3K9me3- and H3K27me3-associated proteinsto E2F-dependent gene promoters^(10, 32, 38, 39). H3K9me3 and H3K27me3are highly enriched on cell cycle gene promoters in ACMs compared toembryonic CMs, with H3K9me3 showing preferential enrichment on G2/M andcytokinesis gene promoters¹⁰. ACM-specific Rb knock out (KO), combinedwith germline deletion of p130, abrogated the heterochromatin formationof cell cycle genes in ACMs¹⁰. ACMs in these mice upregulated cell cyclegenes, including G2/M and cytokinesis genes, which resulted in ACMproliferation. The Rb/p130 KO mice develop heart failure, though it isunclear if it is a result of ACM proliferation, or due to more broadchanges in gene expression profile and loss of globalheterochromatin-organization¹⁰. Rb-family proteins interact with manychromatin-modifiers and transcription factors that also govern geneexpression outside of cell-cycle^(33, 34), making it difficult toattribute changes in the Rb/p130 KO hearts to a single factor orpathway. Specific perturbation of H3K9me3 in vitro by knockdown ofH3K9me3 methyltransferase Suv39h1 resulted in global reduction ofH3K9me3, accompanied with specific re-induction of G2/M and cytokinesisgenes in ACMs, but this was not seen in vivo¹⁰. Knockdown of HP1γ alsospecifically re-induced late cell cycle genes in ACMs, demonstratingthat H3K9me3 and its downstream effector are required for the silencingof these genes in vitro¹⁰, but its physiological role in vivo remainsuncertain.

There remains a need to understand the mechanisms regulating CM cellcycle exit and provide means by which this cell cycle withdrawal can bereversed. There further remains a need for methods of treating ischemicheart disease to reduce the incidence of heart failure and relateddeaths.

SUMMARY OF THE INVENTION

The invention provides an expression vector capable of disrupting thesilencing of cell cycle genes in adult cells, such as adult cardiacmyocytes and other quiescent cells in terminally differentiated tissues.Other examples of quiescent cells and terminally differentiated tissuesin which vectors and methods of the invention can be used to induceproliferation include, but are not limited to, skeletal muscle, neurons,pancreatic islet cells, and hepatocytes. These vectors and methodsprovide tools for regenerative therapy and tissue repair.

In one embodiment, the expression vector comprises: (a) a nucleic acidsequence encoding lysine-specific demethylase 4D (KDM4D); (b) a promoterthat induces or effects overexpression of KDM4D, wherein the promoter isoperably linked to the nucleic acid sequence; and (c) a regulatoryelement that inducibly represses the overexpression of KDM4D.Optionally, the vector further comprises (d) a tissue-specific promoteroperably linked to the nucleic acid sequence. Alternatively,tissue-specific overexpression of KDM4D can be achieved throughselection of a tissue-specific promoter in (b). The KDM4D is capable ofspecifically removing the histone modification H3K9me3 by demethylatingthe lysine residue at position 9 (H3K9) of heterochromatin protein 1(HP1).

In one embodiment, the promoter of (b) is a tissue-specific promoter. Inanother embodiment, separate promoters serve the functions described in(b) and (d) above. Representative examples of tissue-specific promotersinclude, but are not limited to, promoters specific to cardiac tissue,skeletal muscle, neurons, pancreatic islet cells, or hepatocytes. Apromoter that is tissue-specific promotes expression of the gene encodedby the nucleic acid sequence predominantly in the particular tissue. Inone embodiment, the tissue-specific promoter is specific to cardiactissue. An α-myosin heavy chain (αMHC) promoter is one example of acardiac-specific promoter. In another embodiment, the tissue-specificpromoter is specific to liver tissue, or hepatocytes. A CBA promoter isone example of a liver-specific promoter. Other examples oftissue-specific promoters known in the art include the neuron-specificenolase (NSE) and tubulin α1 promoters for neurons, α1-antitrypsin andalbumin (ALB) promoters for hepatocytes, and troponin, CMV, or myosinlight chain-2 (MLC2) for cardiac myocytes.

Representative examples of a regulatory element capable of induciblyrepressing expression (or overexpression) include, but are not limitedto, tetracycline responsive elements. Those skilled in the art willappreciate alternative methods of controlled gene expression that can beadapted for use in a similar manner to regulate the expression of KDM4D,both temporally and histologically. For example, in one embodiment, theregulatory element enables positive regulation of KDM4D expression,while in another embodiment, the regulatory element enables negativeregulation of KDM4D expression. In another example, the regulatoryelement enables tissue-specific and/or condition-specific regulation ofKDM4D expression.

Vectors for use in the methods described herein include viral vectors,as well as non-viral vectors, virus-like particles, bacterial vectors,bacteriophage vectors, and other vectors known in the art, In oneembodiment, the vector is a viral vector. In a particular embodiment,the viral vector is an adeno-associated virus (AAV) vector, or othervector suited for infecting quiescent cells. Representative examples ofan AAV vector include, but are not limited to, AAV6 and AAV9.

The invention also provides a method for inducing proliferation in amammalian cell by reducing H3K9me3 levels in the cell via KDM4D. In oneembodiment, the invention provides a method for inducing tissue-specifichyperplasia in a mammal comprising administering an expression vector asdescribed herein to the mammal. Also provided is a method for inducingcardiac myocyte (CM) hyperplasia in a mammal comprising administering anexpression vector of the invention to the mammal. The invention furtherprovides a method for inducing cardiac myocyte (CM) hyperplasia in amammal. The method comprises grafting CMs to the heart of the mammal,wherein the CMs contain an expression vector of the invention.

The invention additionally provides a method for inducing CM hyperplasiacomprising administering KDM4D to CMs. The KDM4D can be administeredusing a modification of the peptide and/or a delivery means thatprotects the activity of KDM4D. Administration can be oral, intravenous,subcutaneous, or transdermal.

In one embodiment, the invention provides a method of improving organfunction in a mammal comprising grafting cells genetically modified withan expression vector of the invention to the organ. The organ can be,for example, heart, muscle, brain, pancreas, or liver. In oneembodiment, the invention provides a method of improving cardiacfunction in a mammal comprising grafting CMs to the heart of the mammal,wherein the CMs contain an expression vector of the invention. Inanother embodiment, the invention provides a method of improving cardiacfunction in a mammal comprising administering an expression vector ofthe invention to the mammal. Also provided is a method of improvingcardiac function in a mammal comprising administering KDM4D to themammal.

The invention further provides a method of proliferating CM comprisingculturing CM with KDM4D under conditions effective to induce CMhyperplasia. In one embodiment, the CM are adult CM (ACM). In addition,the invention provides a method of promoting cardiac regenerationcomprising reducing lysine 9 of histone H3 (H3K9me3) levels in CMs. Inone embodiment, the reducing comprises administering an expressionvector of the invention to a subject in need of cardiac regeneration. Ina particular embodiment, the expression vector is administered byadministering CMs that contain the expression vector. In anotherembodiment, the reducing comprises administering KDM4D.

The methods of the invention can involve administration to the subjectby any of a variety of means understood by those skilled in the art tobe suitable for particular circumstances. In some embodiments, theadministration is systemic. In other embodiments, the administration isintravenous. In some embodiments, the administration is byintra-myocardial injection. The subject is typically a mammal. In oneembodiment, the mammal is human. In other embodiments, the mammal is aveterinary subject. Examples of veterinary subjects include, but are notlimited to, equine, canine, bovine, porcine, ovine, and feline subjects.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F. Characterization of histone demethylases in development anddisease. (1A-1D) E15.5, P3, P7, and 10 week (adult) CM expression levelsof (1A) cardiac myocyte genes, (1B) cell cycle progression genes, (1C)cell cycle regulators, and (1D) KDM4 H3K9me3-demethylase family membersthrough development. (1E) Gene expression of HDMs in dedifferentiatedmouse ACMs. (1F) KDM4D expression in human ischernic cardiomyopathysample (IHD), expression normalized to GAPDH. Sample Number: (A-D) P0=3.P3=2, P7=4, 10 week=3. (1E) ACM=3, Dedif. ACM=3. (1F) Normal=2, IHD=3,Statistics: (1A-1D) One-way ANOVA/Tukey's test, * P<0.05 vs E15.5, †needed P<0.05 vs P3, ‡ P<0.05 vs P7. (1E-1F) Two-tailed T-test, *P<0.05.

FIGS. 2A-2D. Generation of cardiac myocyte-specific KDM4D model. (2A)Schematic showing breeding strategy resulting in BiTg mice, and KDM4Dinduction in BiTg CMs. (2B) KDM4D transgene expression is robustlyinduced in BiTg ACMs and P14 hearts, fold induction vs. tet control.(2C) BiTg mice display nuclear KDM4D (FLAG-tag) localizationspecifically in CMs. (2D) KDM4D protein induction and global levels ofspecific histone methylations in 9-week ACMs. Sample Number: (2B-2D)Each assay had ≥3 animals per group. Statistics: One-way ANOVA/Tukey'stest, * P<0.05 vs NonTg, † P<0.05 vs tet, ‡ P<0.05 vs tTA.

FIGS. 3A-3C. Gene expression in KDM4D-overexpressing ACMs. (3A) GeneOntology Enrichment scores for “Cellular Process”, and (3B) “Cell CycleProcess”. GO enrichment scores were generated from lists containing allgenes with >3 fold increase in BiTg ACMs at 9 weeks compared controls.(3C) Expression of CM and cell cycle genes in 9-week ACMs, foldinduction vs. NonTg. Sample Number: (3A-3B) Control=2, BiTg=2. (3C)NonTg=3, tet=6, tTA=3, BiTg=5. Statistics: (3A-3B) One-way ANOVA wasused to identify genes with significantly altered expression (P<0.05),Fisher's exact test was used to identify GO terms with significantenrichment scores (P<0.05). (3C) One-way ANOVA/Tukey's test, * P<0.05 vsNonTg, † P<0.05 vs tet, ‡ P<0.05 vs tTA.

FIGS. 4A-4E. Heart mass is increased in KDM4D induced mice. (4A)Representative image showing PFA-fixed BiTg and control hearts at 9weeks, tick marks=1 mm. (4B) Quantification of HW/BW at 9 weeks showingcardiac growth phenotype is specific to BiTg mice. (4C) Quantificationof HW/BW in different ages of mice, normalized to controls for each timepoint. (4D) H&E staining in 9-week NonTg and BiTg hearts. (4E) WGAstaining in 9-week NonTg and BiTg hearts, and 4 weeks post-TAC surgeryin NonTg mice resulting in visible fibrosis. Sample Number: (4A-4B)NonTg=6, tet=11, tTA=9, BiTg=10. (4C) P0, Control=22, BiTg=9; P14,Control=14, BiTg=6; 9 wk Control=26, BiTg=10; 7mo Control=19, BiTg=10.(D-E) Representative images from N≥3 for each group. Statistics: (4B)One-way ANOVA/Tukey's test, * P<0.05 vs NonTg, † P<0.05 vs tet, ‡ P<0.05vs tT.A. (4C) Two-tailed T-test, control vs. BiTg, * P<0.05.

FIGS. 5A-5E. Cardiac myocyte number is increased in BiTg mice. (5A)Left, WGA staining in 9-week NonTg and BiTg PFA-fix hearts, bar=20 μm.Right, quantification of ACM transverse area. (5B) Quantification oflongitudinal area and (5C) length measured in dispersed, isolated 9-wkCMs, with representative images below. (5D) Calculated ACM volume and(5E) ACM number at 9-wks of age. Sample Number: (5A-5E) Each assay had≥3 animals per group. Statistics: (5A) One-way ANOVA/Tukey's test, *P<0.05 vs NonTg, † P<0.05 vs tet, ‡ P<0.05 vs tTA. (5B,5C) Two-tailedT-test, control vs. BiTg, *P<0.05, (5D-5E) The Bootstrap method was usedto compute standard error and Permutation test was used to computep-value, * P<0.05.

FIGS. 6A-6D. Persistent low level cardiac myocyte cell cycle activity inadult BiTg hearts. Mitotic marker phospho-H3 (pH3) staining in NonTg andBiTg heart sections (6A) at P14 and (6B) 9 weeks, bar=40 μm. (6A) Whitearrows point to pH3+ non-CM nuclei, yellow arrowheads point to pH3+ CMnuclei. (6B) Right, high magnification of boxed region, bar=10 μm. (6C)Cell cycling marker Ki67 in 9-week BiTg hearts, bar=10 μm. (6D)Quantification of nuclei number in 7 month old ACMs. Sample Number:(6A-6D) Each assay had animals per group. Statistics: (6D) Two-tailedT-test, control vs. BiTg, * P<0.05.

FIGS. 7A-7D. KDM4D expression induces hyperplastic growth in adult BiTghearts. (7A) Schematic showing usage of doxycycline for temporal controlof CM-specific KDM4D expression in BiTg mice. (7B) Timeline showingprotocol for development-restricted KDM4D expression. (7C) KDM4Dexpression in 9 week or 3 week ventricles of doxycycline (dox) treatedmice, fold induction compared to tet control. (7D) HW/BW at 9-weeks inmouse models where CM-specific KDM4D expression is un-induced (DoxE0-9w), turned off at P14 (Dox 2w-9w), and constitutively expressed (nodox). Sample Number: DoxE0-9w, Control=17, BiTg=4; Dox2w-9w, Control=11,BiTg=8; No dox, Control=26, BiTg=10. Statistics: Two-way ANOVA/Tukey'stest, * P<0.05 vs DoxE0-9w control and BIT g, Dox2w-9w control and BiTg,and no dox control.

FIGS. 8A-8C. Hemodynamic load stimulates hyperplastic growth in BiTghearts. (8A) Representative images of methanol-fixed hearts and (8B)HW/BW quantification of control and BiTg hearts at 10 dayspost-operation, bar=2 mm. (8C) Representative Masson Trichrome stainingof operated mice. Sample Number: Sham, Control=4, BiTg=4; TAC,Control=9, BiTg=8. Statistics: (8B) Two-way ANOVA/Tukey's test, * P<0,05vs Sham-Control, † P<0.05 vs Sham-BiTg, ‡ P<0.05 vs TAC-Control.

FIGS. 9A-9D. Pressure overload stimulates ACM mitotic activity in BiTgmice. (9A) Low and high magnification images of TAC hearts. Bar=40 μm(top) or 20 μm (bottom), white arrows point to pH3+ non-CM nuclei,yellow arrowheads point to pH3+ ACM nuclei. (9B) Quantification of ACMmitotic activity in control and BiTg hearts, 10 days post-operation.(9C) Quantification of ACM transverse area in methanol-fixed hearts, 10days post-operation. (9D) Estimated myocyte cell number. Sample Number:(9A-9D) Sham, Control=3, BiTg=3; TAC, Control=8, BiTg=7. Statistics:(9B,9C) Two-way ANOVA/Tukey's test, * P<0.05 vs Sham-Control, † P<0.05vs Sham-BiTg, ‡ P<0.05 vs TAC-Control. (9D) The Bootstrap method wasused to compute standard error and Permutation test was used to computep-value, * P<0.05 vs control.

FIGS. 10A-10C. KDM4A demethylates H3K9rne3 and H3K36me3 in ACMs. (10A)Timeline showing adenovirus-mediated KDM4A overexpression protocol incultured WT ACMs. (10B) β-galactosidase staining in (top) uninfected and(bottom) lacZ-infected ACMs, showing >80% infection efficiency. (10C)Immunoblot showing KDM4A-expressing ACMs have global reductions inH3K9me3 and H3K36me3, but not in H3K27me3 (Millipore 07449). Lamin A/C(Cell Signaling 47775) and H3 were used as loading controls. SampleNumber: N≥3 for each group.

FIGS. 11A-11B. CM-specific KDM4D transgene expression. (11A) KDM4Dtransgene expression in various BiTg tissue samples at 9 weeks of age,normalized to expression levels in BiTg hearts. (11B) Exogenous KDM4D(FLAG-tag) immunostaining showing lack of expression in non-CM cardiaccells.

FIGS. 12A-12B. Cell Cycle Regulators in BiTg ACM. (12A) Gene expression(RNA-seq, RPKM, fold induction vs. control) of E2F family members in 9week ACMs. (12B) qRT-PCR of cell cycle regulators in 9 week ACMs, foldinduction vs. NonTg. Sample Number: (12A) N=2 per group, (12B) NonTg=3,tet=6, tTA=3, BiTg=5. Statistics: (12A) Two-tailed T-test. * P<0.05.(12B) One-way ANOVA/Tukey's test, * P<0.05 vs NonTg, † P<0.05 vs tet, †P<0.05 vs tTA.

FIGS. 13A-13B. Apoptotic cells are not detected 10 days post-operation.(13A) Representative images of TUNEL staining in vibratome sections.(13B) DNAsel-treated heart sections of adult non-operated mice giverobust nuclear-specific signal, showing our assay is able to detectTUNEL staining. Sample Number: N=2 for each group.

FIGS. 14A-14C. BiTg hearts have increased myocardium and dilated LVchambers. (14A) Representative images of mid-papillary vibratomesections, bar=2 mm. Quantification of (14B) myocardium area and (14C) LVchamber area. Sample Number: N=3 per group. Statistics: Two-wayANOVA/Tukey's test, * P<0.05 vs Sham-Control, † P<0.05 vs Sham-BiTg, ‡P<0.05 vs TAC-Control.

FIGS. 15A-15B. Unique chromatin structure in proliferative CMs. (15A)Immunostaining in embryonic and postnatal wildtype heart sectionsshowing anti-localization of heterochromatin marker H3K9me3 (ActiveMotif, 39161) with euchromatin marker H3K36me3 (Diagenode, C15200183);the change in chromatin organization during postnatal development isalso seen, bar=5 μm. (15B) In BiTg heart sections, pH3+ ACM nuclei(arrowheads) display heterochromatin organization that resemblesembryonic CMs, in contrast to the typical ACM chromatin organization(arrows).

FIG. 16. Neonatal mouse regeneration model. Schematic in upper panelillustrates timeline for creating BiTG mice in which MI occurs at P7 andsacrifice at POD21 for histology and genotyping. Scar detection usesSirius Red+Fast Green staining at 21 days after MI. Fibrotic area isanalyzed as a percentage taken from (the sum of fibrotic area at L600and L800/sum of myocardial area in the LV at L600 and L800)×100.

FIG. 17. KDM4 overexpressing mice have enhanced regeneration post-MI.Histological sections in left panels show Non-BiTG and BiTG samplestaken at indicated L0 to L1000. Bar graphs on right panels show averageand maximum percent fibrotic area for the two groups.

FIGS. 18A-18C. Adult CM-specific KDM4D Expression is sufficient toinduce late cell cycle gene expression in ACMs. (18A) Schematicillustration of doxycycline administration through P21, and later KDM4Doverexpression. (18B) Fold-induction of KDM4D plotted for both tet andBiTg subjects, with doxycycline treatment at E0-P21, or withoutdoxycycline treatment. (18C) Fold-induction of indicated genes forNon-BiTg and BiTg subjects.

FIG. 19. Preliminary data showing adult CM-specific KDM4D expression andcell cycle activity post-MI. Doxycycline chow was administered fromE0-P28. MI occurred at 10 weeks, during period of KDM4D overexpression,and at 14 weeks (30 days post-MI), tissue was examined for phospho-H3,phalloidin, WGA, and Hoechst, comparing control (left panel) and BiTg(right panel).

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the unexpected finding that terminallydifferentiated cells can be induced to proliferate via epigeneticmanipulation. The invention thus provides materials and methods forreversibly inducing proliferation in quiescent cells based on discoveryof the role of H3K9me3 demethylases in regulating ACM cell cycle genesilencing.

Before the discovery of histone demethylases (HDMs)⁴⁰, H3K9me3, andhistone methylation in general, was thought to be a permanent mark⁴¹.However, the dynamic nature of histone methylation is beginning to beappreciated⁴²⁻⁴⁴, though little is known about the functions of HDMs inthe heart. Interestingly, members of the KDM4 family of H3K9me3demethylases are upregulated in several forms of cancer and are thoughtto promote cell proliferation and survival⁴⁵⁻⁴⁸. A member of the KDM4family, KDM4A, has been studied in the heart^(17, 49). CM-specificoverexpression of KDM4A in mice exacerbated TAC-induced hypertrophy andfetal CM gene expression, while CM-specific KDM4A deletion diminishedthe effects of pressure-overload; though neither manipulation had aneffect at baseline⁴⁹. Mechanistic studies demonstrated KDM4A knockdownin neonatal CMs increases H3K9me3 levels at the ANP promoter andmodestly downregulates ANP expression¹⁷. H3K9me3 and HP1 enrichment onthe ANP promoter was reduced in an isolated-working heart model ofelevated preload that induces ANP expression¹⁷. However, KDM4Aexpression and enrichment on the ANP gene promoter were not changed inthis model¹⁷. Thus, it is not clear how KDM4A regulates fetal CM geneexpression in ACMs. Complicating the interpretation of these resultsfurther is the fact that KDM4A has dual-substrate specificity; KDM4A candemethylate repressive H3K9me3, but also activating H3K36me3⁵⁰⁻⁵². Wealso found that global levels of both these modifications were reducedin ACMs with adenovirus-mediated KDM4A overexpression. One KDM4 familymember, KDM4D, has robust and specific H3K9-demethylase activity⁵⁰⁻⁵²,giving it particular usefulness as an experimental tool to study H3K9me3specifically. Until this study, KDM4D has not been explored.

Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

As used herein, “lysine-specific demethylase 4D” or “KDM4D” means aspecific member of the KDM4 family of lysine-specific demethylases thatexhibits demethylase activity specific to the methylated lysine residueat position 9 (H3K9) of heterochromatin protein 1 (HP1). In oneembodiment, the KDM4D has the amino acid sequence shown in SEQ ID NO: 1.The amino acid sequence optionally further includes tags, such as, forexample, a MYC tag and/or a FLAG tag, as shown in SEQ ID NO: 2.

As used herein, “inducibly represses” or “inducible repression” refersto regulation of gene expression whereby expression of the gene can berepressed upon introduction of an inducing condition. The inducingcondition can be administration of or contact with an agent that effectsthe repression. The agent can be a corepressor, such as is found inrepressible gene regulation wherein expression is on except when thecorepressor is present to suppress gene expression. Alternatively, theagent can be an inducer, such as is found in inducible gene regulationwherein expression is off except when the inducer is present to allowfor gene expression.

As used herein, a “regulatory element” refers to an element thatregulates gene expression. The regulatory element may induce or repressgene expression in response to the presence or absence of a condition.

As used herein, a “tetracycline responsive element” refers to aregulatory element that reduces expression from a tet-inducible promoterin the presence of tetracycline or a derivative thereof, e.g.,doxycycline. One example of a tetracycline responsive element is atetracycline-controlled transactivator (tTA), created by fusion of thetetracycline repressor (tetR) with a transcriptional activation domain,such as the C-terminal domain of VP16 of herpes simplex virus (HSV).

The term “nucleic acid” or “polynucleotide” or “oligonucleotide” refersto a sequence of nucleotides, a deoxyribonucleotide or ribonucleotidepolymer in either single- or double-stranded form, and unless otherwiselimited, encompasses known analogs of natural nucleotides that hybridizeto nucleic acids in a manner similar to naturally occurring nucleotides,

The term “primer,” as used herein, means an oligonucleotide designed toflank a region of DNA to be amplified. In a primer pair, one primer iscomplementary to nucleotides present on the sense strand at one end of apolynucleotide fragment to be amplified and another primer iscomplementary to nucleotides present on the antisense strand at theother end of the polynucleotide fragment to be amplified. A primer canhave at least about 11 nucleotides, and preferably, at least about 16nucleotides and no more than about 35 nucleotides. Typically, a primerhas at least about 80% sequence identity, preferably at least about 90%sequence identity with a target polynucleotide to which the primerhybridizes.

As used herein, the term “probe” refers to an oligonucleotide, naturallyor synthetically produced, via recombinant methods or by PCRamplification, that hybridizes to at least part of anotheroligonucleotide of interest. A probe can be single-stranded ordouble-stranded.

As used herein, the term “active fragment” refers to a substantialportion of an oligonucleotide that is capable of performing the samefunction of specifically hybridizing to a target polynucleotide.

As used herein, “hybridizes,” “hybridizing,” and “hybridization” meansthat the oligonucleotide forms a noncovalent interaction with the targetDNA molecule under standard conditions. Standard hybridizing conditionsare those conditions that allow an oligonucleotide probe or primer tohybridize to a target DNA molecule. Such conditions are readilydetermined for an oligonucleotide probe or primer and the target DNAmolecule using techniques well known to those skilled in the art. Thenucleotide sequence of a target polynucleotide is generally a sequencecomplementary to the oligonucleotide primer or probe. The hybridizingoligonucleotide may contain nonhybridizing nucleotides that do notinterfere with forming the noncovalent interaction. The nonhybridizingnucleotides of an oligonucleotide primer or probe may be located at anend of the hybridizing oligonucleotide or within the hybridizingoligonucleotide. Thus, an oligonucleotide probe or primer does not haveto be complementary to all the nucleotides of the target sequence aslong as there is hybridization under standard hybridization conditions.

The term “complement” and “complementary” as used herein, refers to theability of two DNA molecules to base pair with each other, where anadenine on one DNA molecule will base pair to a guanine on a second DNAmolecule and a cytosine on one DNA molecule will base pair to a thymineon a second DNA molecule. Two DNA molecules are complementary to eachother when a nucleotide sequence in one DNA molecule can base pair witha nucleotide sequence in a second DNA molecule. For instance, the twoDNA molecules 5′-ATGC and 5″-GCAT are complementary, and the complementof the DNA molecule 5′-ATGC is 5′-GCAT. The term complement andcomplementary also encompasses two DNA molecules where one DNA moleculecontains at least one nucleotide that will not base pair to at least onenucleotide present on a second DNA molecule. For instance the thirdnucleotide of each of the two DNA molecules 5′-ATTGC and 5′-GCTAT willnot base pair, but these two DNA molecules are complementary as definedherein. Typically two DNA molecules are complementary if they hybridizeunder the standard conditions referred to above. Typically, two DNAmolecules are complementary if they have at least about 80% sequenceidentity, preferably at least about 90% sequence identity.

As used herein, “a” or “an” means at least one, unless clearly indicatedotherwise.

As used herein, to “prevent” or “protect against” a condition or diseasemeans to hinder, reduce or delay the onset or progression of thecondition or disease.

As used herein, the term “isolated” means that a naturally occurring DNAfragment, DNA molecule, coding sequence, or oligonucleotide is removedfrom its natural environment, or is a synthetic molecule or clonedproduct. Preferably, the DNA fragment, DNA molecule, coding sequence, oroligonucleotide is purified, i.e., essentially free from any other DNAfragment, DNA molecule, coding sequence, or oligonucleotide andassociated cellular products or other impurities.

Vectors

In one embodiment, the expression vector comprises: (a) a nucleic acidsequence encoding lysine-specific demethylase 4D (KDM4D); (b) a promoterthat induces or effects overexpression of KDM4D, wherein the promoter isoperably linked to the nucleic acid sequence; and (c) a regulatoryelement that inducibly represses the overexpression of KDM4D.Optionally, the vector further comprises (d) a tissue-specific promoteroperably linked to the nucleic acid sequence. In some embodiments, thetissue-specific overexpression of KDM4D is be achieved through selectionof a tissue-specific promoter in (b). In some embodiments,tissue-specific expression is provided through both (b) and anadditional promoter (d). The KDM4D is capable of specifically removingthe histone modification H3K9me3 by demethylating the lysine residue atposition 9 of histone 3 (H3K9).

While the promoter of (b) can be a tissue-specific promoter, and in someembodiments, separate promoters serve the functions described in (b) and(d) above, the selection of a tissue-specific promoter is designed tooptimize preferential expression in the target tissue while minimizingunintended expression elsewhere. Representative examples oftissue-specific promoters include, but are not limited to, promotersspecific to cardiac tissue (myosin heavy chain, troponin I or T),skeletal muscle (rnyogenein, MyoD, muscle creatine kinase), neurons,pancreatic islet cells, or hepatocytes. A promoter that istissue-specific promotes expression of the gene encoded by the nucleicacid sequence predominantly in the particular tissue. In one embodiment,the tissue-specific promoter is specific to cardiac tissue. An a-myosinheavy chain (αMHC) promoter is one example of a cardiac-specificpromoter. In another embodiment, the tissue-specific promoter isspecific to liver tissue, or hepatocytes. A CBA promoter is one exampleof a liver-specific promoter. Other examples of tissue-specificpromoters known in the art include the neuron-specific enolase (NSE) andtubulin at promoters for neurons, α1-antitrypsin and albumin (ALB)promoters for hepatocytes, and troponin, CMV, or myosin light chain-2(MLC2) for cardiac myocytes.

Representative examples of a regulatory element capable of induciblyrepressing expression (or overexpression) include, but are not limitedto, tetracycline responsive elements and hormone responsive proteins.Those skilled in the art will appreciated alternative methods ofcontrolled gene expression that can be adapted for use in a similarmanner to regulate the expression of KDM4D, both temporally andhistologically. For example, in one embodiment, the regulatory elementenables positive regulation of KDM4D expression, while in anotherembodiment, the regulatory element enables negative regulation of KDM4Dexpression. In another example, the regulatory element enablestissue-specific and/or condition-specific regulation of KDM4Dexpression. While the ability to turn off expression of KDM4D isdesirable, it is not essential to all embodiments. In one embodiment,the invention provides a vector comprising a nucleic acid sequenceencoding lysine-specific demethylase 4D (KDM4D) and a promoter thatinduces or effects overexpression of KDM4D, wherein the promoter isoperably linked to the nucleic acid sequence.

Vectors for use in the methods described herein include viral vectors,as well as non-viral vectors, virus-like particles, bacterial vectors,bacteriophage vectors, and other vectors known in the art. In oneembodiment, the vector is a viral vector. In a particular embodiment,the viral vector is an adeno-associated virus (AAV) vector, or othervector suited for infecting quiescent cells. Representative examples ofan AAV vector include, but are not limited to, AAV6 and AAV9.

KDM4D amino acid sequence (SEQ ID NO: 1):Met E T Met K S K A N C A Q N P N C N I Met I F HP T K E E F N D F D K Y I A Y Met E S Q G A H R AG L A K I I P P K E W K A R E T Y D N I S E I L IA T P L Q Q V A S G R A G V F T Q Y H K K K K AMet T V G E Y R H L A N S K K Y Q T P P H Q N F ED L E R K Y W K N R I Y N S P I Y G A D I S G S LF D E N T K Q W N L G H L G T I Q D L L E K E C GV V I E G V N T P Y L Y F G Met W K T T F A W H TE D Met D L Y S I N Y L H L G E P K T W Y V V P PE H G Q R L E R L A R E L F P G S S R G C G A F LR H K V A L I S P T V L K E N G I P F N R I T Q EA G E F Met V T F P Y G Y H A G F N H G F N C A EA I N F A T P R W I D Y G K Met A S Q C S C G E AR V T F S Met D A F V R I L Q P E R Y D L W K R GQ D R A V V D H Met E P R V P A S Q E L S T Q K EV Q L P R R A A L G L R Q L P S H W A R H S P W PMet A A R S G T R C H T L V C S S L P R Q S A V SG T A T Q P R A A A V H S S K K P S S T P S S T PG P S A Q I I H P S N G R R G R G R P P Q K L R AQ E L T L Q T P A K R P L L A G T T C T A S G P EP E P L P E D G A L Met D K P V P L S P G L Q H PV K A S G C S W A P V P

Optional additional amino acid sequence with myc (underlined) and flag(shaded) tags (SEQ ID NO: 2):

Stop

Compositions & Kits

The invention provides compositions, which can be provided as kitsand/or used for he methods described herein. Compositions of theinvention comprise vectors, nucleic acid molecules, and cells asdescribed herein. Compositions and kits of the invention can includeadditional containers, agents, and materials to facilitate practice ofthe invention.

Methods of the Invention

The invention provides methods for inducing tissue-specific hyperplasiain a mammal comprising administering an expression vector as describedherein to the mammal. The method can be tailored to any organ or tissuein which proliferation or regeneration of quiescent cells is ofinterest. Examples of tissues in which regeneration or proliferation maybe of interest include, but art not limited to, heart, muscle, brain,nervous system, pancreas and liver. Also provided is a method forinducing cardiac myocyte (CM) hyperplasia in a mammal comprisingadministering an expression vector of the invention to the mammal. Theinvention further provides a method for inducing cardiac myocyte (CM)hyperplasia in a mammal. The method comprises grafting CMs to the heartof the mammal, wherein the CMs contain an expression vector of theinvention.

The invention additionally provides a method for inducing CM hyperplasiacomprising administering KDM4D to CMs. The KDM4D can be administeredusing a modification of the peptide and/or a delivery means thatprotects the activity of KDM4D. Administration can be systemic,localized, oral, intravenous, subcutaneous, or transdermal.

In one embodiment, the invention provides a method of improving organfunction in a mammal comprising grafting cells genetically modified withan expression vector of the invention to the organ. The organ can be,for example, heart, muscle, brain, pancreas, or liver. In oneembodiment, the invention provides a method of improving cardiacfunction in a mammal comprising grafting CMs to the heart of the mammal,wherein the CMs contain an expression vector of the invention. Inanother embodiment, the invention provides a method of improving cardiacfunction in a mammal comprising administering an expression vector ofthe invention to the mammal. Also provided is a method of improvingcardiac function in a mammal comprising administering KDM4D to themammal.

The invention further provides a method of proliferating CM comprisingculturing CM with KDM4D under conditions effective to induce CMhyperplasia. In one embodiment, the CM are adult CM (ACM). In addition,the invention provides a method of promoting cardiac regenerationcomprising reducing lysine 9 of histone H3 (H3K9me3) levels in CMs. Inone embodiment, the reducing comprises administering an expressionvector of the invention to a subject in need of cardiac regeneration. Ina particular embodiment, the expression vector is administered byadministering CMs that contain the expression vector. In anotherembodiment, the reducing comprises administering KDM4D.

The methods of the invention can involve administration to the subjectby any of a variety of means understood by those skilled in the art tobe suitable for particular circumstances. In some embodiments, theadministration is systemic. In other embodiments, the administration isintravenous. In some embodiments, the administration is byintra-myocardial injection. The subject is typically a mammal. In oneembodiment, the mammal is human. In other embodiments, the mammal is aveterinary subject. Examples of veterinary subjects include, but are notlimited to, equine, canine, bovine, porcine, ovine, and feline subjects.

EXAMPLES

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

Example 1: Epigenetic Regulation of Cardiac Myocyte Cell Cycle Arrest

This example demonstrates that trimethylation of Lysine 9 of Histone H3(H3K9me3), a histone modification associated with heterochromatin, isrequired for the silencing of cell cycle genes in adult CMs (ACMs). Totest this, we developed a transgenic (BiTg) mouse model where H3K9me3 isspecifically removed by histone demethylase KDM4D in CMs. Loss ofH3K9me3 in CMs disrupts ACM cell cycle gene silencing preferentially andresults in increased CM cycling. Normalized heart mass was increased bypostnatal day 14 (P14) and continued to increase until 9-weeks of age.ACM number, but not size, was significantly increased in BiTg hearts,suggesting CM hyperplasia accounts for the increased heart mass.Challenging H3K9me3-depleted hearts with a hypertrophic growth signalstimulated ACM mitotic activity. Thus, we demonstrated that H3K9me3 isrequired for cell cycle gene silencing in ACMs and depletion of H3K9me3allows hyperplastic growth in vivo.

Methods

Mouse Studies. The αMHC-tTA mice used to control transgene expressionwas generated by the Robbins lab (60). We used the previously publishedresponder construct, which possesses a tetracycline responsive elementupstream of an attenuated αMHC promoter to drive KDM4D expression (60).Plasmid containing FLAG- and MYC-tagged human KDM4D cDNA (origeneRC212600) had a Noll restriction site present in the cDNA sequence,which we destroyed by inducing a silent mutation (Agilent 200521). Theresulting cDNA was subcloned into the responder construct, then freed ofvector backbone, purified, and injected into mouse pronuclei (Universityof Washington transgenic core facility). The resulting tet transgenicwas bred to the oMHC-tTA line to generate the CM-specific KDM4Dinduction model. Littermate controls were used for all experimentsinvolving transgenic mice. TAC and sham operations were performed on 10to 12 week old littermates from breeders backcrossed≥generations to theC57/B6 strain. Mice were anesthetized using ketamine (130 mg/kg i.p.)and xylazine (8.8 mg/kg i.p.) and subjected to transverse aorticconstriction using a 26-gauge needle as described (103).

CM cell isolation and culture. Heparinized mice were euthanized withisoflurane and hearts were extracted and arrested in KB buffer (mmol/L:KCI 20, KH2PO4 10, K+-glutamate 70, MgCl2 1, glucose 25, taurine 20,EGTA 0.5, HEPES 10, 0.1% albumin, pH 7.4 with KOH). For purified ACMpreparations, the aorta was cannulated and the heart was washed withTyrodes solution (pH 7.4, supplemented with 25 uM Blebbistatin −/−) anddigested for 7 minutes with collagenase II (Worthington 4176) andProtease Streptomyces griseus XIV (Sigma P5147) using Langendorfperfusion. Ventricles were dissociated and the resulting cell suspensionwas filtered through a 100 μm mesh. Three rounds of low speedcentrifugation, where ACMs are loosely pelleted and non-CMs insuspension are aspirated, density purify the ACM population, resultingin >90% rod-shaped ACMs. For embryonic and postnatal CM preparations,hearts were washed in Ads buffer (mmol/L: NaCl 116, HEPES 20, NAH2PO410.8, glucose 5,5, KCI 5.4, MgSO4 0.83) and incubated with enzymesolution (Collagenase II, Pancreatin (Sigma P3292)) with rotation. Freedcells were collected into serum (stopping digestion) every 20 minutes,resulting in dissociation of the entire heart within 2 hours. Theresulting cell suspension was fractionated using a percoll (Sigma P4937)gradient, and the CM layer and non-CM layer were each collected. Qualityand purity of CM preparations were verified by immunostaining, flowcytometry, and RNA expression of cell-type-specific markers.

Control ACM and dedifferentiated ACM cDNA was generated as described(57). In brief, ACMs were plated on laminin-coated dishes and culturedwith growth factors for 10-14 days, resulting in a loss of sarcomereorganization and increased CC activity.

RNA isolation and analysis. RNA was isolated from cells and tissue usingTRISOL (Sigma T9424) phenol/chloroform purification, followed by columnpurification with DNase treatment (Qiagen 74004). For human geneexpression studies, normal human heart sample was obtained fromcommercial vendors (Clontech 636532, lot 1206518A; and Agilent 540011,lot 6151000). Ischemic heart disease samples came from consenting malesubjects in their 60's that underwent placement of a left ventricularassist device.

cDNA was synthesized as described in the manufacturers guidelines (Roche04896866001). qPCR was performed using SYBR green (Life Technologies4472908) on a real-time PCR machine (ABI 7900HT). Primers were validatedby standard PCR with electrophoresis to confirm specific target band andlack of primer dimers. qPCR dissociation curves were consistent with asingle specific product. Ct values were assigned using ABI's SDS 2.4software with automated thresholding and baselines. The standard curvemethod or dCt method was used to quantify expression, and expression ofeach gene was normalized by GAPDH. However, in FIG. 1A-D, we present−log(Ct) values. Finding a suitable control gene that is stablyexpressed at different stages in CM development is not trivial (104).Gapdh was the most stably expressed control across all samples comparedto S26 and Rplp0, but compared to normalization by input RNA, Gapdhnormalization resulted in E15.5 CM gene expression being underestimatedby ˜2.5 fold, as Gapdh expression decreases in P3 CMs, then remainsstable; consistent with the high glycolytic activity in fetal hearts(105). Standard Curves were generated using tissue or cells that highlyexpress the indicated gene, resulting in qPCR efficiencies ranging from88-97%. The sequences of primers used are:

Mouse (SEQ ID NOs: 3-50, respectively; individual SEQ ID Nos inparentheses below):

Gapdh (3) F-CCAATGTGTCCGTCGTGGATCT, (4) R-GTTGAAGTCGCAGGAGACAACC; ANP(5) F-AGGATTGGAGCCCAGAGTGGA, (6) R-TGATAGATGAAGGCAGGAAGC; bMHC (7)F-GCGACTCAAAAAGAAGGACTTTG, (8) R-GGCTTGCTCATCCTCAATCC; aMHC (9)F-AGAAGCCCAGCGCTCCCTCA, (10) R-GGGCGTTCTTGGCCTTGCCT; cTNI (11)F-GCAGCCCAGAGGAAACCCAACC, (12) R-AGCCGCATCGCTGCTCTCATC; Ccnd1 (13)F-TGCTGCAAATGGAACTGCTTCTGG, (14) R-TACCATGGAGGGTGGGTTGGAAAT; Ccne1 (15)F-GCTTCGGGTCTGAGTTCCAA, (16) R-GGATGAAGAGCAGGGGTCC; Cdk4 (17)F-GGGACCTGAAGCCAGAGAAC, (18) R-CCACAGAAGAGAGGCTTCCG; Ccnb1 (19)F-GCCTCACAAAGCACATGACTG, (20) R-TCGACAACTTCCGTTAGCCT; Cdk1 (21)F-GGCGAGTTCTTCACAGAGACTTG, (22) R-CCCTATACTCCAGATGTCAACCGG; AurkB (23)F-GCACCTGAAACATCCCAACAT, (24) R-GGTCCGACTCTTCTGCAGTT; Plk1 (25)F-GTATTCCCAAGCACATCAA, (26) R-GTAGCCAGAAGTGAAGAAC; E2F1 (27)F-TGCCAAGAAGTCCAAGAATCA, (28) R-CTGCTGCTCACTCTCCTG; E2F4 (29)F-TGTCCTTGGCAGCACTCA, (30) R-TTCACCACTGTCCTTGTTCTCA; Rb (31)F-CCTGATAACCTTGAACCTGCTTGT, (32) R-GCTGAGGCTGCTTGTGTCT; p130 (33)F-CACCGAACTTATGATGGACAG, (34) R-ATGGCTTCTGCTCTCACT; p107 (35)F-GCAGAGGAGGAGATTGGAACA, (36) R-GCTACAGGCGTGGTGACT; p21 (37)F-GCAGACCAGCCTGACAGATTT, (38) R-CTGACCCACAGCAGAAGAGG; p53 (39)F-CAGTGGGAACCTTCTGGGAC, (40) R-CGCGGATCTTGAGGGTGAAA; KDM4A (41)F-CTGCTAGGGCTTTAGGCTCC, (42) R-TTTGGGAGGAACGACCTTGG; KDM4B (43)F-CAGAGAGCATCACGAGCAGA, (44) R-CTCTTGGGCAGCTCCTCTTC, KDM4C (45)F-GCGGGTTCATGCAAGTTGTT, (46) R-GTTTCAGAGCACCTCCCCTC; KDM4D (endogenous)(47) F-TCTGAGTCTGCCTTCTTCTG, (48) R-GCCAGGGTTCACAAGTCCTGAG;KDM4D (transgene) (49) F-TTGATGGACAAGCCTGTACC, (50)R-TCATTTGCTGCCAGATCCTC.

Mouse: TagMan (Life Technologies) reagents were used for the followinggenes: KDM2B (Mm01194587_m1), KDM4A (Mm00805000_ m1), KDM6B(Mm01332680_m1), KDM8 (Mm00513079_m1), GAPDH (Mm99999915_g1).

Human (SEQ ID NOs: 51-54, respectively; individual SEQ ID Nos inparentheses below):

GAPDH (51) F-CCTCAACGACCACTTTGTCA, (52) R-TTACTCCTTGGAGGCCATGT; KDM4D(53) F-AAGCCCAGCTCAACTCCATC, (54) R-TGTCCATCAAAGCCCCATCC.

RNAseq library construction (IIlumina Tru-Seg) and paired-endRNA-sequencing (AB13730XL) was performed by the Stam Lab's University ofWashington core facility. Read alignment was performed usingBowtie/Cufflinks package. Partek Genomic Suites was used for mRNAquantification, differential expression analysis, and gene ontology.

2-D Echocardiography. Under 0.5% isoflurane, mice EKG and heart functionwas assessed using Visual Sonics Vevo 2100. Parasternal short axisimages at the plane of the papillary muscle were collected in B- andM-Modes. Images were collected with heart rates ranging from 400-500BPMs. Imaging and analysis was performed by a single operator who wasblinded to the genotypes. Quantification of images was performed usingVevo Labs 1.7.0, according to the manufacturer's guidelines.

Protein extraction and Western Blotting. Isolated ACMs were pelleted andresuspended in lysis buffer (0.5% NP-40, 25 mM KCl, 5 mM MgCl2, 10 mMTris-HCl, pH 8.0) and homogenized (Wheaton 358103), releasing solublecytoplasmic proteins. Nuclear-enriched pellets were processed to releasechromatin-associated proteins from DNA; including sonication, MNasetreatment, and addition of 1% SDS, 600 mM NaCl, and 20 mM βME. Thenuclear proteins were quantified using a BOA assay (Thermo Scientific23252) and were loaded on polyacrylamide gels for electrophoresis,subsequently transferred onto PVDF membrane, and probed with theindicated antibodies: KDM4D (Abcam ab93694), H3K9me3 (Abeam ab8898), panH3 (Millipore 05-928). H3K36me3 (Active Motif 61101), H3K9me2 (Millipore07-441), and H4K20me3 (Abcam ab9053). HRP-conjugated secondaryantibodies (Santa Cruz) and ECL-detection (Thermo Scientific 34095) wereused.

Histological studies and quantification CM dimensions and CM number. Forhistological analysis, arrested P14 or 9 week hearts were fixed with 4%PFA, Paraffin sections were stained with H&E, Masson Trichrome, orimmunostained using standard protocol with FLAG (Sigma F7425) α-actinin(Sigma A7811), cardiac Troponin T (Thermo Scientific MS-295-P), Ki67(Abcam ab15580) and phospho-H3 (Abcam ab5176) antibodies, and Hoechst(Life Technologies H3570) to visualize nuclei, images were acquired withconfocal microscopy (Nikon A1R). To assess ACM transverse area, sectionswere stained with Wheat Germ Agglutinin (WGA, Life Technologies W6748),a marker for plasma membrane. Stitched-images of the whole leftventricle were acquired on a Nikon Ti-E scope. We chose several regionsin each section at random, though we excluded large vessels, epicardiumand endocardium, and >1000 cells per animal were analyzed using ImageJ's “analyze particle” function (negative image of WGA stain), resultingin direct measurement of transverse area. For ACM longitudinal area andlength measurements, isolated ACMs were fixed with 4% PFA and imaged.The area and long-axis of hundreds of ACMs for each animal were manuallytraced and quantified using Image J's “measure” function. We calculatedCM volume using the formula: (mean ACM length×mean ACM transverse area).CM number was estimated from the following formula: [mean Heart Volume(Heart mass/1.06, the density of muscle tissue(106))/mean ACMvolume×0.75 (the proportion of adult murine heart volume occupied byCMs(106))] The number of nuclei per ACM was counted manually for >100cells per animal.

Imaging of thick sections. For unambiguous determination of cell type inour phospho-H3 staining assays in operated mice, we developed a methodfor generating, staining, and imaging 100 μm-thick heart sections, whichwill be described in detail in a methodologies article. Briefly, heartswere arrested in KB buffer, perfused with KB, then perfusion fixed withmethanol cooled to −20° C. The hearts were rehydrated in Methanol:PBSgradients (100:0, 80:20, 60:40), then washed with PBS and mounted in 5%low-melt agarose. 100 μm-thick sections were cut from a Leica 1200svibratome and were stained in suspension, with reagents listed above aswell as with Phalloidin (ThermoFisher Scientific A22287). The stainedsections were mounted to glass coverslips coated with 0.01%poly-L-lysine. To increase the transparency of the sections, which isneeded to view the interior of the thick sections, they were cleared:sections were incubated in an isopropanol series (70%, 85%, 95%, 100%)followed by incubations in a 1:2 solution of benzyl alcohol and benzylbenzoate. The samples were prepared, imaged with confocal microscopy,and analyzed by a single operator blinded to the genotypes. Wecalculated the number of pH3+ ACMs using the formula: [(pH3+ ACMnuclei/mm3)/(nuclei#/ACM)/(ACM/mm3)] We note that transverse areameasurements in the sham-operated hearts were 19.2% less than atbaseline, which we attribute to differences in fixation procedure,consistent with other reports comparing cell shrinkage afterformaldehyde or alcohol fixation (107). Because of this, transverse areain all groups was corrected by multiplying by a constant factor of1.192, when calculating ACM number in the post-operation methanol-fixedsamples: [mean Heart Volume (Heart mass/1.06, the density of muscletissue (106))/mean ACM volume (mean ACM baseline length×mean ACMpost-operation transverse area)×0.75 (the proportion of adult murineheart volume occupied by CMs (106))].

Statistics. All results are displayed as mean±standard error of means.Graphpad Prism was used for one-way-ANOVAs and Tukey's post hoc testsperformed on studies comparing more than two groups. Graphpad Prism wasused for two-way-ANOVAs and Tukey's post hoc tests performed on studieswith two independent variables. Microsoft Excel F-test and two-tailedT-test functions were used to analyze studies comparing two groups. Foroutcomes where different basic measurements were combined forcalculations (FIGS. 5, D and E, and FIG. 9D) we used the bootstrapmethod (10,000 bootstrap samples) to compute standard error and thePermutation test (100,000 Monte-Carlo samples) was used to computep-value; with the assumption that ACM transverse area, ACM length, andheart volume are independent variables. For RNA-seg analysis, PartekGenomic Suites was used to perform statistics.

Study approval. All animal studies were performed in accordance with anapproved Institutional Animal Care and Use Committee (IACUC protocol#4290-01), the University of Washington institutional guidelines, andthe National Institute of Health Guide for the Care and Use ofLaboratory Animals. Human ischernic heart disease samples came fromparticipants that gave written and informed consent; the use of humansamples was approved by the University of Washington's InstitutionalReview Board (IRB#35358).

Adenoviral studies. Adenoviruses for KDM4A and LacZ were generatedaccording to manufacturer's guidelines (Agilent 240082). Isolated ACMswere plated on laminin coated wells in M199 medium supplemented with 1×ITS, 1× PS, 5 mM Taurine, 1 mM Na-pyruvate, 5 mM Creatine, 2 mML-carnitine, and 25 mM Blebistatin, with the presence of 5% FBS. After 1hour, media was changed to 2% FBS containing media and 150 moi ofviruses were added. ACMs were maintained in media containing 2% FBSuntil harvesting. Beta-galactosidase staining was performed on 4%PFA-fixed ACMs that were incubated in 5 mM K+ ferri-cyanide, 5 mM K+ferro-cyanide, 2 mM MgCl2, and 1 mg/mL X-gal for 4 hours.

Temporally-controlled KDM4D induction. Doxycycline-containing chow(Harlan TD.00502) was administered ad lib for the indicated times. Notethat the Dox 2 weeks-9 weeks group includes mice that received doxranging from P14-9w to P18-9w.

Myocardium and LV area quantification. Vibratome sections were cut fromthe mid-papillary muscle plane of hearts and imaged. Myocardium area andLV chamber area were manually traced in ImageJ and area was calculatedusing the “measure” tool.

Quantification of apoptosis. Apoptosis was visualized in vibratomesections by using a TUNEL staining kit (Life Technologies C10618)according to the manufacturer's guidelines. Following TUNEL labeling, westained for WGA, Hoechst, and phalloidin, and imaged as described in theprocedures for vibratome sections.

Results Characterization of H3K9me3 Histone Demethylase Expression inCMs

To better understand the role of H3K9me3 in regulating cell cycle geneexpression, we characterized the relationship between cell cycle andH3K9me3-HDM gene expression in CMs through cardiac development (FIG. 1).Developmental changes in CM-specific and cell cycle gene expressionincluded switching of myosin isoforms and dramatic downregulation ofG2/M and cytokinesis genes in ACMs (FIGS. 1, A and B), consistent withprior studies (9,53,54). Cell cycle transcription factor E2F1 wasdownregulated 167-fold in ACMs (P<0.0001, vs. E15.5 CMs), whileexpression of repressive E2F4 remained high (FIG. 1C). Consistent withprior studies in skeletal muscle (35) and CMs (9), we found p107 was theRb-family-member that was expressed specifically in proliferativemyocytes, in contrast to Rb and p130 (FIG. 1C). Expression of KDM4family members followed a similar, though less dramatic, pattern ofexpression as fetal CM, G2/M, and cytokinesis genes, and was moderatelydownregulated after P7 (FIG. 1D), coinciding with loss of CMregenerative potential (2,3). Downregulation of H3K9me3-HDMs in ACMs isconsistent with the increase of global H3K9me3 levels in ACMs comparedto embryonic CMs (9). The low basal level of KDM4D in ACMs is consistentwith other reports of KDM4D expression in tissues with limitedproliferative potential (55,56).

To screen for HDMs that might be involved in CM proliferation, we lookedfor HDMs that were upregulated during CM dedifferentiation, asdedifferentiation appears to be a requisite for CM proliferation in thezebrafish and neonatal mouse heart regeneration models (1-3).Dedifferentiation of mammalian ACMs can be achieved in vitro bylong-term culture with growth factors, resulting in disassembly ofsarcomeres and restoration of proliferative potential (57). From a panelof diverse HDMs, KDM4D was the most highly upregulated (401-fold) duringdedifferentiation (FIG. 1E; P<0.03). Because KDM4A expression iselevated in human hypertrophic cardiomyopathy samples and CM-specificKDM4A overexpression exacerbated hypertrophic growth in mice (49), wewondered if KDM4D was upregulated in human ischemic myocardium. KDM4Dexpression was unchanged in hearts of subjects with ischemiccardiomyopathy (FIG. 1F), consistent with the exceedingly low CMhyperplasia in this setting (58).

Generation of a Transgenic Mouse Model to Deplete H3K9me3 Specificallyin CM

To explore the role of H3K9me3 in ACM cell cycle gene silencing in vivo,we chose to overexpress KDM family member 4D because: 1) KDM4D is themost specific H3K9me3 demethylase (50-52) (FIG. 10), 2) it is expressedin proliferative CMs and elevated in dedifferentiated ACMs (FIGS. 1, Dand E), 3) it is not expressed in cardiomyopathy samples wherehypertrophic growth would predominate (FIG. 1F), 4) it promotesproliferation and survival in non-CMs (46-48), and 5) gain of functionexperiments are less subject to compensation by redundant factors (59).We used a previously characterized tetracycline inducible (tet-off)overexpression model where the tetracycline transactivator (tTA) isexpressed specifically in CMs. (60). We generated a CM-specifictransgenic mouse line containing a MYC-and FLAG-tagged KDM4D cDNAdownstream of a tetracycline responsive promoter, which containstTA-binding sequence in the context of an attenuated-αMHC promoter (60).Breeding heterozygous tTA mice with heterozygous tet-responsive KDM4D(tet) mice yields bi-transgenic (BiTg) mice that constitutively expressKDM4D specifically in CMs (FIG. 2A) as well as single-transgenic (tet ortTA) and non-transgenic (NonTg) controls. In BiTg CMs, the tTA proteinis expressed and binds to the tet-responsive element upstream of KDM4D,inducing KDM4D transgene expression (FIG. 2A). We confirmed that KDM4Dexpression was robustly induced in BiTg hearts at P14 and 9 weeks (FIG.2B). KDM4D transgene expression was not detectable in other organs inBiTg mice or non-CM cardiac cells (FIGS. 11, A and B), with theexception that low levels could be detected in BiTg lungs, consistentwith previous reports using the aMHC promoter (60). Immunofluorescenceimaging in heart sections showed exogenous KDM4D protein wasspecifically expressed and localized in the nuclei of BiTg CMs (FIG.2C). Western blot analysis confirmed KDM4D protein expression and showedglobal H3K9me3 levels were depleted in BiTg ACMs (FIG. 2D). We alsoconfirmed that in contrast to other KDM4 family members (FIG. 10), KDM4Ddemethylase activity is specific to H3K9me3 (50-52) and did notdemethylate H3K9me2 or H3K36me3 in ACMs (FIG. 2D). H4K2Ome3, which hasbeen implicated as a repressive mark that is downstream of H3K9me3 andHP1 (61-63) was unchanged (FIG. 2D); although this does not rule outchanges in methylation levels at specific gene loci.

H3K9me3 is Required for ACM Cell Cycle Gene Silencing in Vivo

To assess the impact of depleting H3K9me3 on global gene expression invivo we performed RNA-sequencing on 9-week ACMs. Control ACM sampleswere grouped since NonTg and single transgenic mice showed nodifferences in gene expression, with the unconstrained slope correlationtest showing R2=0.9764 when comparing the whole-genome transcriptome.RNA-seq analysis revealed that BiTg ACMs had increased expression ofgenes involved in 16 of 138 cellular processes and 16 of 142 cell cycleprocesses (FIG. 3, A and B). Strikingly, cell processes involved in cellcycling were preferentially increased (FIG. 3A). Within cell cycleprocesses, categories involved in the later phases of cell cycle,particularly mitosis showed increased gene expression (FIG. 3B). Weconfirmed increases in G2/M and cytokinesis genes by qRT-PCR (5.8- to21.4-fold, P<0.01) and fetal CM genes were also increased (FIG. 3C).Although the expression of fetal CM genes is frequently associated witha pathologic state, it should be noted that expression of less matureCM-specific genes could also be consistent with proliferation-competentCMs in fetal and neonatal hearts (9,64) (FIGS. 1, A and B). We alsoexamined cell cycle-gene transcriptional regulators (FIGS. 12, A and B)and found that positive regulators of cell cycle progression, E2F1 andE2F2, were highly expressed in BiTg ACMs compared to control ACMs(>12-fold, P<0.03). The repressive E2F members, E2F4-6, were unchanged.Interestingly, p107 was also increased in BiTg ACMs (FIG. 12B),consistent with the E2F/Rb-family expression in proliferative myocytes(FIGS. 1, B and C).

CM-Specific H3K9me3 Depletion Promotes CM Hyperplasia Without AlteringCardiac Function

BiTg mice had visibly larger hearts (FIG. 4A) with a 20.8% increase inheart weight to body weight ratio (HW/BW) at 9 weeks (FIG. 4B;P<0.0001). This increase in HW/BW first became apparent in BiTg mice atP14 (FIG. 4C; 12.9% increase, P<0.001); suggesting KDM4D overexpressionspecifically promoted postnatal cardiac growth. This cardiac enlargementwas not associated with sarcomere disarray, fibrosis or alteration ofvasculature (FIGS. 4, D and E) and there was no increase inextracellular matrix (65) (FIG. 4E). Quantification of ACM transversearea or direct measurements of isolated ACM longitudinal area and lengthdid not reveal differences in dimensions or calculated volumes in BiTgACMs compared to controls (FIG. 5, A-D). Calculated myocyte numbersuggested BiTg hearts had 22% more ACMs compared to controls (FIG. 5E;P<0.03). To determine the longterm effect of H3K9me3-depletion on heartfunction, we performed echocardiography on 7 month old BiTg and controlmice: ejection fractions, fractional shortening, cardiac output, andleft ventricle chamber size were similar in all groups (Table 1). Nosignificant differences in cardiac function or morphology were seen.

TABLE 1 Normal cardiac function and morphology in BiTg mice at 7 months.Echocardiography results in 7 month old mice. NonTg tet tTA BiTg HR(BPM) 440 ± 10  461 ± 16 452 ± 6  404 ± 3_(†‡) EF (%) 74.5 ± 3.7  80.9 ±1.6 83.8 ± 2   80.2 ± 4.6  FS (%) 42.7 ± 3.2  48.9 ± 1.6 52.3 ± 2.2 48.8 ± 4.9  CO (mL/min) 16.4 ± 1.4  20.5 ± 1   17.4 ± 1.5  19.1 ± 0.7 LVEDD(mm) 3.47 ± 0.06  3.61 ± 0.02 3.35 ± 0.15 3.73 ± 0.07 HR: HeartRate, EF: Ejection Fraction, CO: Cardiac Output, LVEDD: Left VentricularEnd-Diastolic Dimension, LV Mass: Left Ventricular Mass. Mean and SEMvalues are shown. Sample Number: N = 3 for each genotype. Statistics:One-way ANOVA/Tukey's test, * P < 0.05 vs NonTg, _(†)P < 0.05 vs tet,_(‡)P < 0.05 vs tTA.

To assess cell cycle activity, we immunostained P14 and 9 weekmyocardial sections for phosphorylated histone H3 serine-10 (pH3), amarker of mitosis. We observed pH3+ CMs only in BiTg mice at both timepoints (FIGS. 6, A and B). Similar trends were seen for the general cellcycle activity marker Ki67 in 9 week hearts (FIG. 6C). Quantification ofthe number of nuclei per ACM at seven months revealed there was anincrease of mononucleated and a decrease in binucleated ACMs in BiTghearts (FIG. 6D). These findings are consistent with a model where theincreased heart mass in BiTg mice was secondary to CM hyperplasia.

Cardiac mass increased up to 9-weeks of age in BiTg mice (FIG. 4C). Cellcycle activity in 9-week BiTg ACMs, though elevated compared to controls(FIGS. 6, B and C), was rare (<2 pH3+ CM; 200× field). To determine ifthe increased mass was related to the normal post-natal hypertrophicgrowth of an increased number of CMs in BiTg hearts or whether there wasongoing CM hyperplasia we utilized doxycycline (Dox) to shut-off KDM4Dexpression (FIG. 7A). We examine heart mass in BiTg mice where KDM4Dexpression was never induced or was induced in utero but suppressed atP14 (FIG. 7B). Dox treatment reduced KDM4D expression in BiTg ventriclesto control levels within one week (FIG. 7C). Adult BiTg mice that hadKDM4D expression suppressed since conception displayed HW/BWs that wereindistinguishable from controls (FIG. 7D). BiTg hearts with constitutiveKDM4D expression were larger at P14 (FIG. 4C; 12.9% increase vs.control; P<0.001). However, when KDM4D expression was turned off at 2weeks, heart size at 9 weeks was less compared to mice with constitutiveKDM4D expression (FIG. 7D; 7.9% vs 20.8%; P<0.05). These findingssuggest that KDM4D overexpression continues to promote additional CMhyperplasia between weeks 2 through 9.

Hypertrophic Signals Stimulate Proliferation of H3K9me3-Depleted ACMs

Although highly upregulated compared to controls, late cell cycle geneexpression in BiTg ACMs is much less than in wildtype embryonic andpostnatal CMs (compare FIG. 1B with FIG. 3C). Also the absolute numberof cycling CMs, while increased in BiTg hearts, was low and normalizedcardiac mass did not increase further between 9 weeks and 7 months (FIG.4C) suggesting CM proliferation is very limited at baseline in adultBiTg hearts. Since there is robust activation of numerous growth factorsignaling pathways post TAC that typically result in hypertrophic growth(9,66), we thought this might be an excellent model to test KDM4D'sability to promote ACM proliferation in vivo. This allowed us todetermine whether a hypertrophic growth signal such as TAC would inducehyperplasia in H3K9me3-depleted ACMs or whether they would undergohypertrophy similar to control hearts. BiTg and control littermatesunderwent sham or TAC surgeries at 11 weeks of age and were examined10-days after surgery. Similar to un-operated mice sham-operated BiTghearts were visibly larger than controls (FIG. 8A). TAC induced a 48.2%increase in HW/BW in BiTg mice compared to a 20.9% in control mice (FIG.8B; P<0.0001). To determine if the TAC-induced heart growth occurredthrough ACM hypertrophy or hyperplasia, we measured ACM transverse area.Sham BiTg ACMs were similar to sham controls (FIG. 5A); however, withTAC BiTg ACM transverse area was 12.5% smaller compared to TAC controlACMs (FIGS. 9, A and C; P<0.05). Since ACM transverse area, but notlength, is increased after TAC (67), we estimated CM number and found a56.6% increase in ACMs in BiTg hearts compared to controls (FIG. 9D,P<0.001). To confirm that BiTg ACMs were cycling after TAC we assayedfor phospho-H3 and observed a 41-fold increase in pH3+ ACMs in BiTg micecompared to control mice (0.77% vs 0.019%; P<0.0001) (FIGS. 9, A and B).This cell cycle activity was not associated with fibrosis (FIG. 8C andFIG. 9A) or increased apoptosis (FIG. 13). To determine if reinductionof CM cell cycling negatively impacted cardiac function, 2-D echoes wereperformed 9 days post-surgery (Table 2). Ejection fraction was decreasedin BiTg mice after TAC along with and an increase in LVEDD. However, thecalculated cardiac output was unchanged, presumably due to the chambersize in BiTg hearts (FIG. 14).

TABLE 2 Cardiac function and morphology in TAC-operated mice.Echocardiography results in 12 week old mice, 10 days post-operation,Sham TAC Control BiTg Control BiTg HR (BPM) 428 ± 17  416 ± 7.6 430 ± 11406 ± 10  EF (%) 71.1 ± 4.2 72.4 ± 3.7 75.5 ± 4.4 51.2 ± 3.7* FS (%)39.5 ± 3.5 40.8 ± 3.4 43.7 ± 3.5 25.9 ± 2.3* CO (mL/min) 11.6 ± 2.4 12.4± 1.5  9.8 ± 0.9 10.9 ± 0.7  LVEDD(mm)  3.04 ± 0.14  3.19 ± 0.11  2.83 ±0.09  3.57 ± 0.10* HR: Heart Rate, EF: Ejection Fraction, CO: CardiacOutput, LVEDD: Left Ventricular End-Diastolic Dimension, LV Mass: LeftVentricular Mass. Mean and SEM values are shown. Sample Number: Sham,Control = 4, BiTg = 4; TAC, Control = 9, BiTg = 8. Statistics:Two-tailed T-test, control vs. BiTg, *P < 0.05.

Discussion

Since the discovery that neonatal mammalian hearts can regenerate by CMproliferation (2,3) and that ACMs retain some, though very limited,capacity to divide (7), interest in the regulation of ACM cell cycle hasbeen reignited (4,69-71). Many strategies that can promote proliferationin mammalian neonatal CMs have been ineffective in ACMs (72,73)highlighting the fact that strong barriers to proliferation exist inACMs. Recently, epicardial paracrine factor FSTL1 (70), miR-15inhibition (3), and the NRG1 co-receptor Erbb2 (74) were suggested topromote ACM proliferation, though the molecular mechanisms and relevanttargets in ACMs remain elusive. We previously found that theheterochromatin marker H3K9me3 was enriched on G2!M and cytokinesisgenes in ACMs compared to fetal CMs implicating this mark as a barrierto ACM proliferation (9). This example characterizes the expression ofH3K9me3-demethylases in development, dedifferentiation, and disease andshows that KDM4D was expressed in fetal CMs and was the primaryH3K9me3-specific demethylase upregulated in dedifferentiating ACMs.CM-specific KDM4D overexpression depleted H3K9me3 specifically and ledto increases in ACM expression of G2/M and cytokinesis genes, cardiacmass, ACM number, and ACM mitotic activity.

The renewed interest in CM proliferation has also called attention tothe need of improved methodologies for detecting ACM proliferation (75).The standard indicator of ACM proliferation has been indirect in situhistology of phase-specific or general cell cycle markers. However, ithas been suggested these results are often equivocal because it can bedifficult to determine if the marker is present in a CM or non-CM(75,76). Most studies are performed in cardiac sections less than 10 μmthick, which is significantly thinner than even the shortest axis ofmammalian ACMs, and are further confounded by the dense myocardialvasculature and non-CMs that are the majority of cardiac cells (77). Weaddressed this limitation by developing a novel sample preparation andimaging technique that yields high-resolution 3D image reconstructionsof cleared thick sections with several layers of whole AGMs, allowingunambiguous identification of mitotic ACMs in KDM4D overexpressing mice(FIG. 9). Our methods corroborate findings from in vivocumulative-proliferation-labeling in CM lineage-tracing models(multi-isotope-mass-spectrometry (7), mosaic analysis with doublemarkers (78), and multi-color clonal assays (76)) and supports theemerging consensus that ACM proliferation is rare and difficult tostimulate (75). Despite the fact that late cell cycle gene expressionwas markedly increased at 9 weeks in H3K9me3-depleted CMs (FIG. 3C)their expression levels were much lower when compared to neonatal CMs(FIG. 1B). As well, the vast majority of BiTg ACMs appeared to exit thecell cycle by P14 (FIG. 6A) and the difference in HW/BW compared tocontrols did not appear to increase further after 9 weeks (FIG. 4C).This suggests mechanisms, in addition to H3K9me3, prevent proliferationin ACMs.

This example confirms that H3K9me3 is required for ACM cell cycle genesilencing in vivo but that H3K9me3 is not sufficient by itself toexplain the stable silencing of cell cycle genes in ACMs since it wasmaintained on G2/M and cytokinesis genes even after they werederepressed by Rb/p130 double knockout (9). In that study, HP1γ, whosechromodomain specifically binds H3K9me3, was displaced from late cellcycle gene promoters. This study suggested that stable silencing of cellcycle genes in ACMs required recruitment of HP1γ and that H3K9me3,though required, was not sufficient to silence genes (9). Since Rb andH3K9me3 are both required for HP1 binding in many systems it is possiblethat KDM4D overexpression derepressed late cell cycle genes in ACMs byremoving HP1γ's binding-substrate from chromatin. Regardless, since Rband H3K9me3 are present on numerous genes it remains unclear why latecell cycle genes are preferentially derepressed. It may perhaps berelated to particular combinations of E2F- and Rb-family members formingdistinct complexes and targeting specific subsets of genes (33,34). InBiTg ACMs, this example showed that the E2F/Rb-family members mostincreased by H3K9me3-depletion were E2F1 and p107 (FIG. 12), which arethe same E2F/Rb-family members that are specifically expressed inproliferative embryonic and neonatal CMs (FIG. 1C) (9). Consistent withHP1 losing the ability to target E2F-dependent genes in BiTg ACMs, p107,in contrast to Rb, has not been shown to bind HP1 (39). In agreement, Rbhas additional protein-binding and phospho-regulated domains not foundin p107, and deletion of p107 has not shown the hyperplasia phenotypeseen with Rb loss of function (79;80). Thus, selective increases inE2F/Rb-family expression levels and differential recruitment of HP1 mayexplain the preferential increase of late cell cycle genes in BiTg ACMs.

This example demonstrated that ACMs can tolerate moderate levels of G2/Mand cytokinesis gene expression without deleterious effects on heartfunction (FIG. 3 and Table 1). This is similar to other models oflimited ACM cycling (76), but contrasts with our previous findings wheredisrupting heterochromatin formation and inducing cell cycle reentry inACMs was associated with decreased heart function (9). A fundamentaldifference between the KDM4D mice and that model is that KDM4Doverexpression specifically targets one methylation pathway, whereasRb/p130 KO likely disrupted multiple epigenetic modifications (H3K9me3,H3K27me3, and H4K20me3) (9,32,33). This may explain why genes involvedin promoting all phases of cell cycle were upregulated in Rb/p130 KO,while H3K9me3- or HP1γ-specific disruption leads to preferentialincrease of G2/M and cytokinesis gene expression(9) (FIG. 3). Consistentwith this notion, H3K9me3-depleted chromatin in KDM4D mice maintainedits global structure including heterochromatin unlike the Rb/p130 modelwith the exception of cycling pH3+ ACMs (FIG. 15). Thus, repressivemethylations have overlapping roles in maintaining global chromatinstructure in ACMs consistent with reports of H3K9me3-depletion in othercell types (27,81,82).

We have suggested that there is a transition in heterochromatinstructure during postnatal differentiation in ACMs (9) from limitedheterochromatin organized into many small foci within the nucleus ofembryonic CM, to few, large foci with additional heterochromatin at thenuclear lamina in postnatal CM nuclei (FIG. 15A). We found that the pH3+ACMs in BiTg mice had a gross chromatin structure similar to that ofproliferative embryonic CMs (FIG. 15B). The relationship betweenhigher-order chromatin structure and gene-specific regulation is notclear, though studies suggest they act independently (27,82).Interestingly, H3K9me3 and H3K9me3-associated proteins also regulatecell cycle via transcription-independent mechanisms involving changes inglobal chromatin structure required for DNA synthesis and mitosis(42,83). Not surprisingly, pH3, the hallmark of mitotic activity, isinhibited by trimethylation of the adjacent amino acid residue H3K9 anddouble knockout of H3K9me3 methyltransferases Suv39h1/2 resulted inincreased pH3+ mouse embryonic fibroblasts (84,85), suggesting thatglobal H3K9me3-depletion facilitates mitotic activity. Though severalmechanisms may contribute to KDM4D-mediated ACM cell cycle activity, wehave provided evidence that supports a model where cell cycle genesilencing is prevented by depleting H3K9me3, but additional repressorsappear to prevent robust cell cycle activation in the absence of growthstimulation.

The results can be compared to findings in studies of another major cellproliferation regulation signaling pathway, the organ-size-controllingHippo/Yap pathway. This pathway has been intensely studied with severalCM-specific loss of function and gain of function mouse models throughCM development and adulthood (71,93). Though Yap1 gain of function inadults increased ACM proliferation, the levels were 20-fold less thanNonTg neonatal CMs (76). The authors postulated Yap activation alone isinsufficient to overcome the multiple barriers blocking ACMproliferation. In several other systems Yap signaling fails to driveproliferation in the absence of E2F signaling (94-96). Interestingly,informatics and chromatin immunoprecipitation sequencing approachesfound E2F- and Yap-binding sites neighbor each other on many cell cyclegene promoters (95-97), which suggests E2F and Yap might be parallelpathways. Indeed, in liver regeneration models enhanced E2F activationby triple knockout of the Rb-family members resulted in cellproliferation; however the increased proliferation declined over timedue to dampening of Yap signaling (97). Forced Yap1 activation orreducing liver size by partial hepatectomy allowed the E2F-mediatedincreases in proliferation to persist. This suggests that the intrinsicHippo/Yap pathway has a remarkable ability to sense and regulate normalorgan size and that E2F-mediated increases in proliferation can beaugmented by growth stimulation or Yap signaling. This is consistentwith our finding that hemodynamic load, which increases active Yaplevels (98) stimulated dramatic proliferation even in quiescent adultKDM4D hearts. Interestingly, endogenous Yap1 protein is expressed highlyin postnatal CMs and is maintained at 8-weeks but lost by 20-weeks (99),which may account for the increases in HW/BW leveling out by 9-weeks inour model. The synergism of E2F and Yap activation is also consistentwith a model of multiple blocks to ACM proliferation. Future studiesexamining the connectivity of the E2F/Rb-family and Hippo/Yap signalingpathways in CM cell cycle may clarify if H3K9me3 regulation of cellcycle genes is strictly E2F dependent. The relative importance of thesepathways is speculative but growth signals that typically induce ACMhypertrophy caused re-induction of ACM proliferation in bothH3K9me3-depleted ACMs and previously reported Yap1 mice (76). AlthoughNonTg ACMs in the present example and these YAP-activated models hadsimilar, very limited mitotic activity after growth stimulus (˜0.02%(76) vs 0.019% FIG. 9B) we found substantially more mitotic BiTg ACMsafter TAC than seen in Yap1-activated ACMs after MI (0.77% FIG. 9B vs0.07% (76)). Whether all BiTg ACMs retained proliferative potential orthere is a subset of highly proliferative ACMs is unclear but if pH3signal is present for 3 hours of a 22 hour cell cycle (102) it wouldsuggest that 5.7% of BiTg ACMs were actively cycling 10 days after TAC.

In conclusion, CM-specific KDM4D induction and the subsequentH3K9me3-depletion is sufficient to maintain proliferation competence inACMs. These results further the understanding of how cardiac growth isregulated and identify a new role for H3K9me3 and common effectorpathway for regulation of CM cell cycling. The fact that KDM4Doverexpression did not affect normal heart function but allowedhyperplasia in response to hypertrophic signals supports the use ofKDM4D to improve the regenerative response in clinical settings. Thisstrategy is very amenable to gene therapies with localized andtemporally controlled KDM4D expression and provides regenerative cardiactherapies.

Example 2: Neonatal Mouse Regeneration Model

An animal model for regeneration of heart tissue is illustrated in FIG.16. Using this model, mice overexpressing KDM4D show enhancedregeneration after myocardial infarct (MI) per histological analysis, asillustrated in FIG. 17. A statistically significant (p<0.05) reductionin both average fibrotic area and maximum fibrotic area was observed inKDM4D overexpressing mice compared to control mice at 21 days after MI.These data demonstrate the utility of KDM4D for regenerative therapy ofcardiac tissues.

Example 3: Gene Therapy for Heart Regeneration

Vectors can be constructed for implementation and assessment of cardiacregeneration using gene therapy. The vectors can be constructed usingAAV viruses, such as AAV6 or AAV9 or other hybrid AAV serotypes offeringspecificity for cardiac myocytes. A Troponin promoter is used forCM-specific expression. The AAV-KDM4D vector (expressing SEQ ID NO: 1and optionally SEQ ID NO: 2) is injected into cardiac tissue at, orshortly after, the time of myocardial infarct. Assessment of heartfunction by echocardiography (ECHO) and cardiac magnetic resonance (CMR)is performed at subsequent time intervals to monitor recovery andregeneration. At the conclusion of the study, infarct size and ACMproliferation are assessed.

In one version of the construct, KDM4D can be shut off after a few weeksof regeneration to return KDM4D levels back to baseline, once lost CMshave been repopulated. Those skilled in the art will appreciateadjustments to the protocol that can be made to allow for alternativevectors and/or promoters that can achieve the same effect, as well as toallow for appropriate timing of the period of treatment before returningKDM4D levels back to baseline, taking into account individualcircumstances such as the extent of damage and/or patientresponsiveness.

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Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to describemore fully the state of the art to which this invention pertains.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

1.-20. (canceled)
 21. A method for inducing cardiomyocyte (CM)hyperplasia comprising delivering a transgene encoding exogenouslysine-specific demethylase 4D (KDM4D) to CMs.
 22. The method of claim21, wherein the KDM4D comprises an amino acid sequence of SEQ ID NO:1.23. The method of claim 21, wherein the method comprises contacting theCMs with an expression vector comprising: (a) a nucleic acid sequenceencoding KDM4D; (b) a cardiac-specific promoter capable of effectingoverexpression of KDM4D only in cardiac tissue, wherein the promoter isoperably linked to the nucleic acid sequence; and (c) a regulatoryelement that inducibly represses the overexpression of KDM4D.
 24. Themethod of claim 23, wherein the expression vector is a viral vector thatinfects quiescent cells.
 25. The method of claim 24, wherein the viralvector is an adeno-associated virus (AAV) vector.
 26. The method ofclaim 25, wherein the AAV vector is AAV6 or AAV9.
 27. The method ofclaim 21, wherein the CMs are adult CMs (ACMs).
 28. A method ofimproving cardiac function in a mammal comprising delivering a transgeneencoding exogenous KDM4D to the mammal.
 29. A method of proliferatingCMs comprising culturing CMs with a transgene encoding exogenous KDM4Dunder conditions effective to induce CM hyperplasia.
 30. A method ofpromoting cardiac regeneration in a subject in need thereof comprisingreducing lysine 9 of histone H3 (H3K9me3) levels in CMs.
 31. The methodof claim 30, wherein the reducing comprises contacting the CMs with anexpression vector comprising: (a) a nucleic acid sequence encodingKDM4D; (b) a cardiac-specific promoter capable of effectingoverexpression of KDM4D only in cardiac tissue, wherein the promoter isoperably linked to the nucleic acid sequence; and (c) a regulatoryelement that inducibly represses the overexpression of KDM4D.
 32. Themethod of claim 31, wherein the KDM4D comprises an amino acid sequenceof SEQ ID NO:1.
 33. The method of claim 31, wherein the expressionvector is a viral vector that infects quiescent cells.
 34. The method ofclaim 33, wherein the viral vector is an adeno-associated virus (AAV)vector.
 35. The method of claim 34, wherein the AAV vector is AAV6 orAAV9.
 36. The method of claim 31, wherein the expression vector isdelivered by delivering CMs that contain the expression vector.
 37. Themethod of claim 30, wherein the reducing comprises delivering atransgene encoding exogenous KDM4D.
 38. The method of claim 37, whereindelivery is systemic.
 39. The method of claim 37, wherein delivery isintravenous.
 40. The method of claim 37, wherein delivery is byintra-myocardial injection.